Preparation of Hop Acids and Their Derivatives

ABSTRACT

A method for preparing a hop acid mixture having an enantiomeric excess of a (+)-tetrahydro-α-acid is disclosed. In the method, a racemate of a tetrahydro-α-acid is contacted with an amine to form a precipitate having an enantiomeric excess of the (+)-tetrahydro-α-acid. A method for preparing a hop acid is also disclosed. In the method, a racemate of a tetrahydro-α-acid is contacted with an amine to form a precipitate comprising a (+)-tetrahydro-α-acid, and the (+)-tetrahydro-α-acid is isomerized to a hop acid selected from the group consisting of (+)-trans-tetrahydro-iso-α-acids, (−)-cis-tetrahydro-iso-α-acids, and mixtures thereof, and reduced to (+)-trans-hexahydroiso-α-acids and (−)-cis-hexahydroiso-α-acids. An additive for flavoring a malt beverage is also disclosed. The additive includes a bittering agent selected from the group consisting of (+)-trans-tetrahydro-iso-α-acids, (−)-cis-tetrahydro-iso-α-acids, (+)-trans-hexahydroiso-α-acids, (−)-cis-hexahydroiso-α-acids, and mixtures thereof.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 13/112,770 filed May 20, 2011, which claims priority from U.S.Patent Application No. 61/347,201 filed May 21, 2010.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

Not Applicable.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to novel hop acid compounds that provide improvedflavor, foam, and antimicrobial contributions in malt beverages such asbeer and active ingredients for health supplements. In particular, theinvention relates to methods for preparing a hop acid mixture having anenantiomeric excess of a (+)-tetrahydro-α-acid, methods for preparing(+)-tetrahydro-α-acids that can be isomerized to(+)-trans-tetrahydro-iso-α-acids and (−)-cis-tetrahydro-iso-α-acids, andreduced to (+)-trans-hexahydroiso-α-acids and(−)-cis-hexahydroiso-α-acids and malt beverage bittering agentsincluding the (+)-trans-tetrahydro-iso-α-acids,(−)-cis-tetrahydro-iso-α-acids, (+)-trans-hexahydroiso-α-acids,(−)-cis-hexahydroiso-α-acids, or mixtures thereof.

2. Description of the Related Art

Chiral recognition of substances, i.e. the ability to distinguish amolecular structure from its mirror image, is one of the most importantand widespread principles of biological activity. The first molecularevent in odor perception is the interaction of an odorant with areceptor. As olfactory receptors have been identified as proteins, i.e.chiral molecules, this interaction should also be enantioselective,meaning that odor receptors should react differently with the twoenantiomeric forms of a chiral odorant, leading to differences in odorstrength and/or quality. Discrepant enantiomer effects arewell-established, with numerous examples in taste perception. Forexample, limonene is present in both orange and lemon peels andresponsible for their different odor characteristics because orangecontains the right-handed (+) molecule while lemon contains theleft-handed (−) molecule; (S)-(+)-carvone is a molecule withcaraway-like odor while its mirror image molecule (R)-(−)-carvone has aspearmint odor. Linalool is one of main key hop flavor components inbeer, which optical isomers have great impact on the character of hoppyflavor. (−)-Linalool is perceived with woody, lavender-like aroma, whileits mirror image molecule, (+)-linalool, has sweet and citrus-likearoma.

Tetrahydroiso-α-acids (including three major analogs oftetrahydroisocohumulone, tetrahydrohumulone, and tetrahydroadhumulone)have shown more benefits in brewing than their analogous of iso-α-acids,ρ-iso-α-acids, and hexahydroiso-α-acids. Tetrahydroiso-α-acids impartthe most bitter intensity, provide more light stability and flavorstability, enhance more foam, and exhibit stronger antimicrobialactivity than the other hop bittering compounds in beer.Tetrahydroiso-α-acids are prepared from either α-acids (including threemajor analogs of cohumulone, n-humulone, and adhumulone) or β-acids(including three major analogs of colupulone, n-lupulone, andadlupulone) (See, P. Ting & H. Goldstein, J. Am. Soc. Brew. Chem.54(2):103-109, 1996). From the α-acids (humulones), sequentialhydrogenation and isomerization reactions or reversed isomerization andhydrogenation reactions of α-acids are involved as shown in FIG. 1,wherein R=CH₂CH(CH₃)₂ for n-humulone, R=CH(CH₃)₂ for cohumulone, andR=CH(CH₃)CH₂CH₃ for adhumulone. From the β-acids (lupulones), multiplereactions are involved including a sequentialhydrogenolysis/hydrogenation reaction of β-acids, oxidation reaction ofthe hydrogenated desoxy-α-acids and then an isomerization reaction oftetrahydro-α-acids as shown in FIG. 1, wherein R=CH₂CH(CH₃)₂ forn-lupulone, R=CH(CH₃)₂ for colupulone, and R=CH(CH₃)CH₂CH₃ foradlupulone.

Both methods produce identical molecules of tetrahydroiso-α-acids, butonly different from their stereoisomers. Tetrahydroiso-α-acids preparedfrom α-acids are optically active compounds, or enantiomers, due to thenatural structure of α-acids (asymmetry molecules) (see, D. DeKeukeleire and M. Verzele, J. Inst. Brewing, 76:265, 1970). However,tetrahydroiso-α-acids prepared from β-acids (no asymmetry molecules) area racemic mixture (containing pairs of mirror image molecules or equalopposite enantiomers) with no optical activity (see, Patrick L. Ting andHenry Goldstein, J. Am. Soc. Brew. Chem. 54(2):103-109, 1996).

The molecular perception of stereochemistry of tetrahydroiso-α-acids andhexahydroiso-α-acids prepared from either α-acids or β-acids is veryimportant because of their potential flavor, foam, antimicrobialcontributions in beer as well as important ingredients fornutraceuticals and functional food (see U.S. Pat. No. 7,270,835).However, the stereochemistry and physiological properties (chiralrecognition) have not been investigated and reported fortetrahydroiso-α-acids prepared from β-acids.

Therefore, there still exists a need for tetrahydroiso-α-acid compoundsthat improve flavor, foam, and antimicrobial contributions in maltbeverages such as beer.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a method for preparing a hop acidmixture having an enantiomeric excess of a (+)-tetrahydro-α-acid. In themethod, a racemate of a tetrahydro-α-acid is contacted with an amine toform a precipitate having an enantiomeric excess of the(+)-tetrahydro-α-acid. The precipitate can be treated to prepare a solidhaving an enantiomeric excess of the (+)-tetrahydro-α-acid of greaterthan 50%, or more preferably greater than 80%. The amine can be a chiralamine such as (1S, 2R)-(−)-cis-1-amino-2-indanol. The racemate of thetetrahydro-α-acid can be prepared by hydrogenating a β-acid to prepare adesoxy-α-acid, and oxidizing and isomerizing the hydrogenateddesoxy-α-acid to prepare the racemate of the tetrahydro-α-acid. In oneversion of the method, the β-acid is colupulone, and the desoxy-α-acidis tetrahydrodesoxycohumulone. The tetrahydro-α-acid can be selectedfrom tetrahydrohumulone, tetrahydrocohumulone, and tetrahydroadhumulone.In one version of the method, the precipitate is separated from afiltrate, and the precipitate is treated such that the precipitate hasan enantiomeric excess of the (+)-tetrahydro-α-acid of greater than 80%,and the filtrate is treated such that a solid recovered from thefiltrate has an enantiomeric excess of a (−)-tetrahydro-α-acid ofgreater than 80%. A reversed solid-liquid process is possible using adifferent amine.

In another aspect, the invention provides a method for preparing a hopacid. In the method, a racemate of a tetrahydro-α-acid is contacted withan amine to form a precipitate comprising a (+)-tetrahydro-α-acid; andthe (+)-tetrahydro-α-acid is isomerized to a hop acid selected from thegroup consisting of (+)-trans-tetrahydro-iso-α-acids,(−)-cis-tetrahydro-iso-α-acids, and mixtures thereof. The(+)-trans-tetrahydro-iso-α-acid can be selected from(+)-trans-tetrahydro-iso-humulone, (+)-trans-tetrahydro-iso-cohumulone,and (+)-trans-tetrahydro-iso-adhumulone, and the(−)-cis-tetrahydro-iso-α-acid can be selected from(−)-cis-tetrahydro-iso-humulone, (−)-cis-tetrahydro-iso-cohumulone, and(−)-cis-tetrahydro-iso-adhumulone. The amine can be a chiral amine suchas (1S,2R)-(−)-cis-1-amino-2-indanol. In one version of the method, theracemate of the tetrahydro-α-acid can be prepared by hydrogenating aβ-acid to prepare a desoxy-α-acid, and oxidizing and isomerizing thehydrogenated desoxy-α-acid to prepare the racemate of thetetrahydro-α-acid.

In another aspect, the invention provides a method for preparing groupof novel hop acids, (+)-tetrahydro-α-acids isomerized and reduced to agroup selected from the group consisting of (+)-hexahydroiso-α-acids,(−)-hexahydroiso-α-acids, and mixtures thereof.

In yet another aspect, the invention provides an additive for flavoringa malt beverage, wherein the additive includes a bittering agentselected from the group consisting of (+)-trans-tetrahydro-iso-α-acids,a (−)-cis-tetrahydro-iso-α-acids, and mixtures thereof. The(+)-trans-tetrahydro-iso-α-acid can be selected from the groupconsisting of (+)-trans-tetrahydro-iso-humulone,(+)-trans-tetrahydro-iso-cohumulone, and(+)-trans-tetrahydro-iso-adhumulone, and the(−)-cis-tetrahydro-iso-α-acid can be selected from the group consistingof (−)-cis-tetrahydro-iso-humulone, (−)-cis-tetrahydro-iso-cohumulone,and (−)-cis-tetrahydro-iso-adhumulone.

In still another aspect, the invention provides novel ingredients fornutraceutical and functional foods, wherein the active ingredientsincludes a bittering agent selected from the group consisting of(+)-tetrahydro-α-acids, (+)-trans-tetrahydroiso-α-acids,(−)-cis-tetrahydroiso-α-acids, (+)-trans-hexahydroiso-α-acids,(−)-cis-hexahydroiso-α-acids, and mixtures thereof.

In yet another aspect, the invention provides a method for preparing ahop acid mixture. The method comprises contacting a racemate oftetrahydroiso-α-acids with a chiral amine to form a hop acid complex asa precipitate or in solution such that the hop acid complex has anenantiomeric excess of (+)-tetrahydroiso-α-acids. The enantiomericexcess of the resolved (+)-tetrahydroiso-α-acids can be greater than50%, preferably greater than 60%, preferably greater than 70%,preferably greater than 80%, and preferably greater than 90%. Theresolved tetrahydroiso-α-acids can be enantiomerically pure. Theresolved (+)-tetrahydroiso-α-acids can be reduced to a hop acid selectedfrom the group consisting of (+)-trans-hexahydro-iso-α-acids,(−)-cis-hexahydro-iso-α-acids, and mixtures thereof.

In still another aspect, the invention provides a method for preparing ahop acid mixture. The method comprises resolving a racemate oftetrahydroiso-α-acids with a chiral column chromatography to separate anenantiomeric excess of (+)-tetrahydroiso-α-acids. The enantiomericexcess of the resolved (+)-tetrahydroiso-α-acids can be greater than50%, preferably greater than 60%, preferably greater than 70%,preferably greater than 80%, and preferably greater than 90%. Theresolved tetrahydroiso-α-acids can be enantiomerically pure. Theresolved (+)-tetrahydroiso-α-acids can be reduced to a hop acid selectedfrom the group consisting of (+)-trans-hexahydro-iso-α-acids,(−)-cis-hexahydro-iso-α-acids, and mixtures thereof.

In yet another aspect, the invention provides an additive for flavoringa malt beverage wherein the additive comprises a bittering agentselected from the group consisting of (+)-trans-tetrahydroiso-α-acids,(−)-cis-tetrahydroiso-α-acids, (+)-trans-hexahydroiso-α-acids,(−)-cis-hexahydroiso-α-acids, and mixtures thereof. In still anotheraspect, the invention provides a malt beverage including the additivewherein the bittering agent is present in the malt beverage at a levelof 1 ppm to 100 ppm.

In still another aspect, the invention provides an active ingredient fora health supplement wherein the ingredient comprises a hop acid selectedfrom the group consisting of (+)-tetrahydro-α-acids,(+)-trans-tetrahydro-iso-α-acids, (−)-cis-tetrahydro-iso-α-acids,(+)-trans-hexahydro-iso-α-acids, (−)-cis-hexahydroiso-α-acids, andmixtures thereof.

These and other features, aspects, and advantages of the presentinvention will become better understood upon consideration of thefollowing detailed description, drawings and appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a scheme of tetrahydroiso-α-acids preparation from eitherα-acids or β-acids.

FIG. 2 shows the analytical HPLC resolution of (±)-tetrahydrocohumuloneby a 250×4.6mm β-Cyclobond column with 75% CH₃CN+25% of 1% acetic acidin 20% CH₃OH/H₂O) at 280 nm and 1.2 ml/min.

FIG. 3 is a diagram of the dynamic resolution of (±)-THCO.

FIG. 4 is a plot of circular dichroism of (±),(+), and (−)-THCO.

FIG. 5 shows the chiral HPLC separation of isomerized (+) and (−)-THCO(top) and two CD spectra of (+) vs. (−)-trans-THICO (bottom).

DETAILED DESCRIPTION OF THE INVENTION

In one example method of invention, dynamic resolution of a racemictetrahydro-α-acid mixture has been achieved. Suitable solvents for theresolution can be selected such that at least some of the desireddiastereomeric solid precipitates in the solvent and the other member ofthe pair remains dissolved in the solution. Non-limiting examplesolvents include substituted or unsubstituted aliphatic or alicyclichydrocarbons. Some preferred solvents are hexane, cyclohexane andtoluene. The solvents and the temperature of the dynamic resolution willvary with the particular hop acid subjected to the resolution.

Precipitation of the desired diastereomeric solid can be achieved with achiral amine. A preferred amine is one that allows formation of a pairof diastereomeric solids, one member of the pair of diastereomers beingat least partially insoluble in the solvent system of the process. Apreferred amine is one that allows formation of a pair of diastereomers,one member of the pair being preferentially a precipitate under thereaction conditions. The precipitate can be crystalline ornon-crystalline. The chiral amine can be, for example,(1S,2R)-(+)-cis-1-amino-2-indanol. Other chiral amines can be expectedto be useful in effecting the resolution of the hop acid.

The less soluble diastereomeric salt from the reaction can be isolated,for example, by filtration, centrifugation, or decantation. Forinstance, the reaction mixture is cooled to room temperature and theresulting precipitate is recovered by filtration. The filter cakecontaining the product can be washed with a washing solvent such as analiphatic hydrocarbon (e.g., hexane). Once isolated, the precipitateddiastereomeric salt can be liberated from its complexed chiral amine byreaction with a suitably strong acid. Non-limiting example acids includesulfuric acid, phosphoric acid and hydrochloric acid. The diastereomericcompound that remains in solution in the filtrate can also be isolatedwith an acid.

Dynamic resolution of the racemate of a tetrahydro-α-acid or racemate ofa tetrahydroiso-α-acid in accordance with the invention can produce anenantiomeric excess of the one of the resolved tetrahydro-α-acids ortetrahydroiso-α-acids of greater than 50%, preferably greater than 60%,preferably greater than 70%, preferably greater than 80%, and preferablygreater than 90%. The resolved tetrahydro-α-acid or(+)-tetrahydroiso-α-acid can be enantiomerically pure. In one form, theresolved tetrahydro-α-acid is (+)-tetrahydrocohumulone.

The resolved tetrahydro-α-acids can be isomerized totetrahydro-iso-α-acids by boiling in a suitable solvent such as anethanol-water mixture, optionally in the presence of a catalyst such asa calcium or magnesium salt. Other isomerization techniques can be used.The tetrahydroiso-α-acid compounds can provide improved flavor, foam,and antimicrobial contributions when added to malt beverages such asbeer. In one form, the tetrahydro-iso-α-acid is(+)-trans-tetrahydro-iso-cohumulone or(−)-cis-tetrahydro-iso-cohumulone. The resolved tetrahydro-α-acids canbe reduced to (+)-trans-hexahydroiso-α-acids and(−)-cis-hexahydroiso-α-acids.

A variety of optical isomers have been described as having differentodor qualities and/or different odor intensities. Such considerationsprompted the following experimental study, which aimed to resolve gramquantities of each enantiomer of (±)-tetrahydrocohumulone and to assesstheir isomerized enantiomers for bitterness, foam quality, andantimicrobial activity. The following Examples are presented forpurposes of illustration and not of limitation.

EXAMPLES

HPLC of (±)-tetrahydrocohumulone (THCO) and (+/−) and(−/+)-cis/trans-tetrahydroisocohumulones (THICO)

A 5 μm 250×4.6 mm Cyclobond I 2000 column (Advanced SeparationTechnologies Inc.) was used. To resolve (±)-THCO, an isocratic mixtureof 25% A (CH₃CN) and 75% B (1% acetic acid +20% methanol/H₂O) was usedas the mobile phase at a flow rate 1.2 mL/min and detection at 280 nm.Two enantiomers, (−) and (+)-THCO, were eluted, respectively in FIG. 2.(−)-THCO was identified by the hydrogenated α-acids standard. To resolve(±)-cis and (±)-trans-THICO, an isocratic mixture of 35% A (CH₃CN) and65% B (0.01M sodium citrate +20% methanol/H₂O) was used as the mobilephase at a flow rate 1.2 mL/min and detection at 254 nm. The elutionorder was (−)-trans, (+)-trans, (−)-cis, and (+)-cis-THICO identified bythe retention times of (+)-cis-THICO and (−)-trans-THICO standards asshown in FIG. 5.

Preparation of Tetrahydrodesoxycohumulone

To a solution of 50 g hexane-crystallized colupulone (0.125 moles) in250 mL of ethanol was added 10 mL of concentrated sulfuric acid and 5 gof 5% Pd/C catalyst. The mixture was stirred and hydrogenated under 10psig hydrogen gas in an autoclave. The hydrogenation reaction wascompleted after 30 min. at 35-50° C. The vessel was purged with nitrogenand the mixture was filtered to give a clear yellow solution oftetrahydrodesoxycohumulone, used directly in the next step.

Oxidation With Peracetic Acid of Tetrahydrodesoxycohumulone to(±)-tetrahydrocohumulone (THCO)

To the above solution was added 23.75 g of 40% peracetic acid (0.125moles) slowly in a three-neck round bottom flask which was equipped witha thermometer, a condenser, and additional funnel. After addition, thereaction was heated to 50-60° C. for 1 hour and allowed to cool to roomtemperature. About 100 mL tap water was added and stirred for 1 hour.The ethanol was recovered under vacuum and 200 mL hexane was added tosolubilize the THCO in aqueous solution. After a phase separation, thehexane solution was washed with tap water twice to afford 35 g of THCO.

Resolution of (±)-tetrahydrocohumulone (THCO)

To a solution of 11 g of (±)-tetrahydrocohumulone (THCO) (0.031 moles),20 mL toluene and 300 mL cyclohexane was added 5.5 g of (1S,2R)-(−)-cis-1-amino-2-indanol (Al) (0.037 moles) in 50 mL cyclohexane.The solution was boiled for 15 min. and allowed to cool at roomtemperature. A yellow solid was crystallized and filtered from thesolution. The yellow solid was mixed with 100 mL hexane and 100 mL of 2NHCl. The hexane phase was washed three times with water. After dryingwith anhydrous magnesium sulfate, the solvent was removed under vacuumto yield 4.65 g of (+)-THCO and confirmed by a chiral HPLC (β-Cyclobondcolumn) with 90% enantiomeric excess (e.e.). The filtrate was acidifiedwith 100 mL of 2N HCl and washed with water three times. After dryingwith anhydrous magnesium sulfate, the solvent was removed by vacuum toafford 5.3 g of 86% e.e. (−)-THCO and confirmed by β-Cyclobond HPLC.

Isomerization of (+), (−), and (±)-tetrahydrocohumulone (THCO) tocis/trans-tetrahydroisocohumulone

To 2.5 g of THCO (7.1 mmoles) and 50 mL ethanol was stirred and heatedwith 0.3 g NaOH and 43 mg of magnesium sulfate. The isomerizationreaction was refluxed for 2 hours and allowed to cool at roomtemperature. The resulted solution was acidified by 2N HCl and ethanolwas recovered by vacuum. The resulted oil was extracted by 50 mL hexaneand two phases were separated. The hexane phase was dried by anhydrousmagnesium sulfate and the solvent was removed by vacuum to yieldenantiomers and a racemate of cis/trans-tetrahydroisocohumulone (THICO).

Conclusion

(1S, 2R)-(−)-cis-1-Amino-2-indano is an unequivocal chiral reagent forresolving racemic hop acids. It reacts with (±)-tetrahydrocohumulones toselectively produce a crystal form of (+)-tetrahydrocohumulone(THCO)/(1S, 2R)-(−)-cis-1-amino-2-indano from the solution of (−)-THCOand (1S, 2R)-(−)-cis-1-amino-2-indano. It is a dynamic process that caneasily process several grams of (±)-tetrahydro-α-acids for anyapplication. The (+)-THCO is a novel bittering precursor as well as itsderivatives of (−)-cis/(+)-trans-tetrahydroisocohumulone (THICO). On theother hand, its counterpart, (−)-THCO, is identical to the hydrogenated(−)-cohumulone (a nature α-acid). Three THICO molecules can beenantioselectively distinguished by our receptors, leading to differentodor intensities and odor qualities. THICO II from its (+)-THCO have themost bitterness and longest lingering and (±)-THICO III have milder andsmooth bitterness than THICO I from (−)-THICO. The foam situation ismore complicated by findings of enantioselective effects of each chiralisomer with damaged conformation of LTP (a foam protein) during thekettle boiling. Three THICO I, II, and III molecules demonstrate thesame antibacterial effectiveness using the minimum inhibitoryconcentration (MIC) and the bacterial zone of inhibition (BZI) tests onPediococcus damnosus and Lactobacillus brevis. It clearly indicates thatthe enantioselectively antibacterial interactions between THICO I, II,III and the microbial do not occur.

Results and Discussion

The molecular perception of stereochemistry of tetrahydroiso-α-acidsprepared from either α-acids or β-acids is very important because oftheir potential flavor, foam, antimicrobial contributions in beer.Verzele and De Keukeleire (D. De Keukeleire and M. Verzele, J. Inst.Brewing, 76:265, 1970; “Chemistry and analysis of Hop and Beer BitterAcids”, M. Verzele and D. De Keukeleire, Elsevier, 1991) haveestablished the R-configuration and (−)-optical rotation of α-acids withan asymmetric center at C-6 and also determined the optical propertiesof their isomerized derivatives denoted as (+)-cis-iso-α-acids and(−)-trans-iso-α-acids. The hydrogenation of either α-acids orisomerized-α-acids retains the chirality, in other words, no opticalproperty is changed. On the other hand, the β-acids preparedtetrahydroiso-α-acids are a racemic mixture consisting of two pairs of(+/−)-cis/trans- and (−/+)-cis/trans-isomers from the isomerization of aracemic mixture of (±)-tetrahydro-α-acids. Because the β-acids aredissymmetric or achiral compounds, the hydrogenolysis/hydrogenation ofβ-acids produce planar molecules, tetrahydro desoxy-α-acids with a C2symmetry. At the oxidation step, a chiral center at C-6 is introduced togenerate a pair of (±)-tetrahydro-α-acids as shown in FIG. 1.

Resolution by Liquid Chromatography

Ting and Goldstein confirmed the optical rotations of the hydrogenatediso-α-acids as (+)-cis- and (−)-trans-tetrahydroiso-α-acids. (Patrick L.Ting and Henry Goldstein, J. Am. Soc. Brew. Chem. 54(2):103-109, 1996),while a zero value of optical rotations of (±)-cis- and(±)-trans-tetrahydroiso-α-acids is obtained from the β-acidspreparation. Ting and Goldstein successfully resolved and assigned(±)-tetrahydro-α-acids using a combination of a semi-preparative C-18column and an analytical Cyclobond HPLC column (β-Cyclodextrin bonded on5 μ silica gel, a chiral phase column). The column was also used toresolve most of (+) and (−)-enantiomers and diastereomers of total(±)-tetrahydroiso-α-acids.

To evaluate the properties of each enantiomer of tetrahydroiso-α-acidsin beer, a gram quantity of substances is needed. Due to the complexcompositions of hop bittering compounds (containing at least of 12compounds with 3 major analogs and 2 diastereoisomers and 2enantiomers), a strategy of simplifying the resolution process isstarting with colupulone, one component of β-acids, to produce(±)-tetrahydrocohumulone. Resolution of (±)-tetrahydrocohumulone (THCO),bittering precursors, should be less complicated than their isomerized(±)-cis- and (±)-trans-tetrahydroisocohumulones (THICO).

An analytical chiral HPLC (high pressure liquid chromatography) column(β-Cyclobond) was used to analyze and identify the resolved compounds asshown in FIG. 2. In FIG. 2, (±)-THCO is well-resolved into (−)-THCOidentified by authentic (−)-tetrahydro-α-acids and eluted before(+)-THCO. Since a chiral liquid chromatography (LC) was a prevalenttechnique of resolving enantiomers, two β-Cyclobond 10×2″ and 20×2″columns simulated to the analytical conditions were used to separate theracemic mixture of (±)-THCO at milligrams to gram quantities. Theresolution of (±)-THCO was poor and ineffective.

Resolution by Dynamic Crystallization

Alternatively, using an (−)-alkaloid, (1S, 2R)-(−)-cis-1-amino-2-indanol(Al) to react with (±)-THCO becomes a dynamic resolution technique(Chemical & Engineering News, Sep. 9, 2002). Two diastereomeric saltswere formed; one, (+)-THCO/(−)-alkaloid, was crystallized out and leftone, (−)-THCO/(−)-alkaloid in the solution as shown in FIG. 3. Afteracidification, it regenerated high optically pure (+) and (−)-THCO,separately, as well as more than gram quantities yield sufficient toperform various tests. A chiral HPLC and CD (Circular Dichroism)confirmed the optical purity and optical spectrum of resolved (+) and(−)-THCO vs. (±)-THCO (FIG. 4). The identity of (−)-THCO were confirmedby comparison of the retention time of chiral HPLC and CD spectrum withthe hydrogenated α-acids. The (+)-THCO is a novel bittering precursorhaving an opposite CD spectrum as (−)-THCO which is a hydrogenatednatural α-acid. The isomerization of (+)-THCO produced two novel(−)-cis/(+)-trans-tetrahydroisocohumulone (THICO) in opposite to(−)-THCO produced (+)-cis/(−)-trans-THICO identical to the hydrogenatednatural iso-α-acids. FIG. 5 shows a chiral HPLC separation/resolution of(+/−) and (−/+)-cis- and trans-THICO and two CD spectra of (+)- and(−)-trans-THICO. The bitter perception, foam quality, and antimicrobialactivity of three molecules and their derivatives of(+/−)-cis/trans-THICO (THICO I), (−/+)-cis/trans-THICO (THICO II), and(±)-cis/trans-THICO (THICO III) were investigated, respectively.

Bitterness Perception

An aqueous 5% v/v ethanol/H₂O solution was spiked with 6 ppm of THICO I,II, and III. The bitterness of three molecular perceptions is summarizedin Table 1. It indicates that the bitterness intensity is II>III>I andthe bitter perception of III is smooth and milder than the others.

TABLE 1 Bitterness of three THICO I, II, and III in 5% ethanol/H₂O THICOI (natural) THICO II (novel) THICO III (racemic) Less bitter at back ofMost bitter and Stronger than I, but similar, tongue, harsh, slightlingering, smooth, milder, clean lingering astringent, bitter inbitterness whole mouth

Two sets of unhopped lagers (A and B) were spiked with 6 ppm and 13 ppmof THICO I, II, and III, respectively. A C-18 reversed phase HPLCanalysis of cis/trans-THICO present in each beer is shown in Table 2.

TABLE 2 HPLC analysis of cis/trans-tetrahydroisocohumulones (THICO) inBeer THICO I (ppm) THICO II (ppm) THICO III (ppm) A 5.6 6.0 6.0 B 13.712.2 13.8

Sensory evaluation indicated that beer with THCO II was noted as havingthe strongest initial bitterness and lingered the longest. The other twobeers were noted as being similar with the THICO I having a little moreinitial bitterness and the bitterness in the THICO III beer diminishedquickly. In set B, THICO II beer had a strong initial bitterness thatincreased (described as late bitterness) and also lingered. The othertwo beers were noted as being similar with initial intense bitternessthat diminished slowly with slight lingering bitterness. It indicatesthat our odor receptors can differentiate two enantiomeric THICO I andII, leading to differences in bitter strength and quality.

Foam

One major factor of beer foam is an interaction of a lipid transferprotein (LTP) from barley with the hop bittering compounds. (see, L.Lusk, H. Goldstein, D. Ryder, J. Amer. Soc. Brew. Chem. 53(3):93-103,1995). Tetrahydroiso-α-acids interact preferentially with LTP due totheir greater hydrophobicity. (see, K. Takeshi and T. Shellhammer, J.Agric. Food Chem., 2008, 56 (18), pp 8629-8634). The Nibem and half-lifefoam test of three bittering molecules in beers in set A do not show anysignificant differences (see Table 3). Discrepancy of theenantioselective effects between enantiomers of THICO and LPT is notclear in the beer foam formation. It might be due to disruption of theconformation of LPT which has been known to be damaged after long kettleboiling (Sandra N. E. Van Nierop, David E. Evans, Barry C. Axcell, IanC. Cantrell, and Marina Rautenbach, J. Agric. Food Chem., 2004, 52 (10),pp 3120-3129; E. N. Clare Mill, Chunli Gao, Peter J. Wilde,

Neil M. Rigby, Ramani Wijesinha-Bettonis, Victoria E. Johnson, Lorna J.Smith and

Alan R. Mackie, Biochemistry, 2009, 48 (51), pp 1208-12088).

TABLE 3 Results of beer foam and bittering molecules Nibem 30 sec.Half-Life THICO I 255 4.7 THICO II 259 5.0 THICO III 246 5.3

Antimicrobial Activity of THICO I, II, III and Minimum InhibitoryConcentration (MIC) and Bacterial Zone of Inhibition (BZI)

The antimicrobial effect of three molecules was tested on Pediococcusdamnosus and Lactobacillus brevis with two methods (MIC and BZI). MICwas determined based on the concentration at which no bacteria weredetected in the modified BMB without Tween 80 culture medium. The resultis summarized in Table 4 and the MIC is 16 ppm for both THICO I and II.The average diameters of the zones of bacterial inhibition in UniversalBeer Agar (UBA) produced by the filter paper disks immersed in 4000 ppmof THICO I, II, and III in 70% ethanol/water are shown in Table 5. Zonediameters increased with the same rates for three molecules on twodifferent organisms (Pediococcus damnosus and Lactobacillus brevis)indicate that all molecules have the same antibacterial effectiveness.No enantioselective antibacterial interactions between THICO I, II, IIIand the microbial occur.

TABLE 4 Minimum inhibitory concentration of THICO I and II Pediococcusdamnosus Minimum Inhibitory Concentration of Hop Acids in 70% Ethanolppm 128 64 32 16 8 4 2 1 0.5 0 THICO I − − − +/− + + + + + + THICO II −− − +/− + + + + + + + = Growth of beer spoilage bacteria − = No growthof beer spoilage bacteria +/− = Partial inhibition of bacterial growth

TABLE 5 Antimicrobial effect of THICO I, II, and III on bacterialDiameter of Bacterial Zone of Inhibition (mm) Pediococcus damnosusLactobacillus brevis Control 0 0 THICO I 11.5 26 THICO II 12 26.5 THICOIII 12 22.5

Thus, in the present invention, resolution of a racemic(±)-tetrahydrocohumulone (or tetrahydro-α-acid) and their isomerizedtetrahydroisocohumulones (or tetrahydroiso-α-acid) has been achieved ingram quantity by a dynamic crystallization with (1S,2R)-(−)-1-amino-2-indanol. The resolved (+)-tetrahydrocohumulone (THCO)is a novel bittering precursor while the (−)-THCO is identical to thehydrogenated (−)-cohumulone (a natural α-acid). Both enantiomers areisomerized to the same molecular structures, but with opposite opticalrotations. The (+)-THCO is converted into two novel bitteringdiastereomers, (−)-cis- and (+)-trans- tetrahydro isocohumulone (THICOII) while (−)-THCO is converted into (+)-cis- and (−)-trans-THICO (THICOI) identical to the hydrogenated cis and trans-isocohumulone (a naturaliso-α-acid).

Sensory indicates that the bitter intensity of three molecules is THICOII>(±)-THICO III>THICO I. The perception of (±)-THICO III is smooth,clean and milder than I and II. In the foam situation, it seems noapparent foam quality differences among three molecular beers. In otherwords, no clear discrepancy of enantioselective effects among threemolecules and lipid transfer protein (LTP) is found. It may be due todestruction of LTP conformation during long kettle boiling.

The minimum inhibitory concentration (MIC) of THICO I and II is similarat 16 ppm against Pediococcus damnosus. Zone diameters increased withthe same rates for three THICO I, II, and III molecules on two differentorganisms (Pediococcus damnosus and Lactobacillus brevis) indicate thatall exhibit the same antibacterial effectiveness or no enantioselectiveantibacterial effect among THICO I, II, III and the microbial.

Although the invention has been described in considerable detail withreference to certain embodiments, one skilled in the art will appreciatethat the present invention can be practiced by other than the describedembodiments, which have been presented for purposes of illustration andnot of limitation. Therefore, the scope of the appended claims shouldnot be limited to the description of the embodiments contained herein.

What is claimed is:
 1. An additive for flavoring a malt beverage, theadditive comprising: a bittering agent selected from the groupconsisting of (+)-trans-tetrahydroiso-α-acids,(−)-cis-tetrahydroiso-α-acids, (+)-trans-hexahydroiso-α-acids,(−)-cis-hexahydroiso-α-acids, and mixtures thereof.
 2. The additive ofclaim 1 wherein: the bittering agent is (+)-trans-tetrahydroiso-α-acid.3. The additive of claim 1 wherein: the bittering agent is(−)-cis-tetrahydroiso-α-acid.
 4. The additive of claim 1 wherein: thebittering agent is (+)-trans-hexahydroiso-α-acid.
 5. The additive ofclaim 1 wherein: the bittering agent is (−)-cis-hexahydroiso-α-acid. 6.A malt beverage including the additive of claim 1, wherein the bitteringagent is present in the malt beverage at a level of 1 ppm to 100 ppm. 7.A malt beverage including the additive of claim 2, wherein the bitteringagent is present in the malt beverage at a level of 1 ppm to 100 ppm. 8.A malt beverage including the additive of claim 3, wherein the bitteringagent is present in the malt beverage at a level of 1 ppm to 100 ppm. 9.A malt beverage including the additive of claim 4, wherein the bitteringagent is present in the malt beverage at a level of 1 ppm to 100 ppm.10. A malt beverage including the additive of claim 5, wherein thebittering agent is present in the malt beverage at a level of 1 ppm to100 ppm.
 11. A method for flavoring a malt beverage, the methodcomprising: adding to the malt beverage a bittering agent selected fromthe group consisting of (+)-trans-tetrahydroiso-α-acids,(−)-cis-tetrahydroiso-α-acids, (+)-trans-hexahydroiso-α-acids,(−)-cis-hexahydroiso-α-acids, and mixtures thereof.
 12. The method ofclaim 11 wherein: the bittering agent is (+)-trans-tetrahydroiso-α-acid.13. The method of claim 11 wherein: the bittering agent is(−)-cis-tetrahydroiso-α-acid.
 14. The method of claim 11 wherein: thebittering agent is (+)-trans-hexahydroiso-α-acid.
 15. The method ofclaim 11 wherein: the bittering agent is (−)-cis-hexahydroiso-α-acid.16. The method of claim 12, wherein the bittering agent is added to themalt beverage at a level of 1 ppm to 100 ppm.
 17. The method of claim13, wherein the bittering agent is added to the malt beverage at a levelof 1 ppm to 100 ppm.
 18. The method of claim 14, wherein the bitteringagent is added to the malt beverage at a level of 1 ppm to 100 ppm. 19.The method of claim 15, wherein the bittering agent is added to the maltbeverage at a level of 1 ppm to 100 ppm.